U.S. patent number 5,213,669 [Application Number 07/829,638] was granted by the patent office on 1993-05-25 for capillary column containing a dynamically cross-linked composition and method of use.
This patent grant is currently assigned to Beckman Instruments, Inc.. Invention is credited to Andras Guttman.
United States Patent |
5,213,669 |
Guttman |
May 25, 1993 |
Capillary column containing a dynamically cross-linked composition
and method of use
Abstract
Disclosed herein is a capillary column containing a dynamically
cross-linked composition and method of use. In a particularly
preferred embodiment, the dynamically cross-linked composition
comprises: 1.0% polyethylene oxide; 1.0% polyethylene glycol; 1.0%
ethylene glycol; 100 mM TRIS-CHES buffer; and 0.1% sodium dodecyl
sulphate, where the pH of the composition is between about 8.0 and
about 9.0 and the viscosity of the composition is less than about
500 centipoise. The disclosed compositions can be used for the
analysis of surfactant:proteinaceous material complexes and the
generation of calibration curves over an extensive range of
molecular weights, using capillary electrophoretic techniques. The
disclosed compositions are particularly suited for capillary
electrophoretic systems having UV-based detection.
Inventors: |
Guttman; Andras (Palo Alto,
CA) |
Assignee: |
Beckman Instruments, Inc.
(Fullerton, CA)
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Family
ID: |
25255092 |
Appl.
No.: |
07/829,638 |
Filed: |
January 31, 1992 |
Current U.S.
Class: |
204/452; 204/455;
204/601; 204/605 |
Current CPC
Class: |
G01N
27/44704 (20130101); G01N 27/44747 (20130101) |
Current International
Class: |
G01N
27/447 (20060101); G01N 027/26 (); B01D
057/02 () |
Field of
Search: |
;204/180.1,299R |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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90301508.9 |
|
Aug 1991 |
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EP |
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WO9111709 |
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Aug 1991 |
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WO |
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Other References
Bode, H-J. "SDA-Polyethyleneglycol Electrophoresis: A Possible
Alternative to SDS-Polyacrylamide Gel Electrophoresis" 65/1:56-58
FEBS Letters (1976). .
Widhalm, A. et al., "Capillary Zone Electrophoresis with a linear
non-crosslinked polyacrylamide gel: Separation of proteins
according to molecular mass," J. Chron. 549: 446-451 (1991). .
Tsuji, K. "High-Performance Capillary Electrophoresis of Proteins.
Sodium dodecyl sulphate-polyacrylamide gel-filled capillary column
for the determination of recombinant biotechnology-derived
proteins." J. Chron. 550: 823-830 (1991)..
|
Primary Examiner: Valentine; Donald R.
Assistant Examiner: Starsiak, Jr.; John S.
Attorney, Agent or Firm: May; William H. Harder; P. R
Burgoon, Jr.; Richard P.
Claims
What is claimed is:
1. A capillary column containing a dynamically cross-linked
composition comprising:
a) a capillary column having an interior cavity defined by a wall
with an inner surface;
b) a dynamically cross-linked composition filling said interior
cavity, said composition comprising:
i) between about 0.1% and about 1.5% polyethylene oxide;
ii) between about 0.0% and less than about 2.0% polyethylene
glycol;
iii) between about 0.0% and about 2.0% of a surfactant;
iv) between about 0.0M and about 1.0M of a pH buffer; and
v) between about 0.0% and about 99% of a polyol; where the pH of
the composition is between about 2.0 and about 10.0 and the
viscosity of the composition is less than about 4,000
centipoise.
2. The capillary column of claim 1 further comprising a layer of a
coating material on said inner surface of said wall.
3. The capillary column of claim 1 wherein said capillary is
constructed of a material selected from the group consisting of
glass, alumina, beryllia, fused silica and TEFLON.
4. The capillary column of claim 1 wherein said capillary is
constructed of fused silica.
5. The capillary column of claim 1 wherein the internal diameter of
said capillary is between about 2 .mu.m and about 2000 .mu.m.
6. The capillary column of claim 1 wherein said composition
comprises about 1.0% polyethylene oxide.
7. The capillary column of claim 1 wherein said composition
comprises about 1.0% polyethylene glycol.
8. The capillary column of claim 1 wherein said surfactant is
selected from the group consisting of sodium-dodecyl sulphate,
decyl-sulphate, polyoxyethylene ethers, polyoxyethylenesorbitans,
deoxycholate, cetyltrimethylammonium bromide and cetylpyridinium
chloride.
9. The capillary column of claim 1 wherein said surfactant is
sodium dodecyl sulphate.
10. The capillary column of claim 9 wherein said composition
comprises about 0.1% sodium dodecyl sulphate.
11. The capillary column of claim 1 wherein said pH buffer is
ultra-violet light transparent.
12. The capillary column of claim 1 wherein said pH buffer
comprises zwitterionic buffers.
13. The capillary column of claim 1 wherein said pH buffer is
selected from the group consisting of 2-(N-morpholine)
ethanesulfonic acid, N-(2-acetamido) iminodiacetic acid,
piperazine-N,N'-bis(2-ethanesulfonic acid, N
(2-acetamido)-2-aminoethanesulfonic acid, (2-aminoethyl)
trimethyl-ammonium chloride hydrochloride,
N,N-bis(2-hydroxy-ethyl)-2-aminoethane sulfonic acid,
N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid,
tris-hydroxymethyl amino methane,
N-tris(hydroxyl-methyl)methylglycine,
N,N-bis(2-hydroxyethyl)-glycine, 2-(N-cyclohexylamino)
ethane-sulfonic acid and mixtures of the foregoing.
14. The capillary column of claim 1 wherein said pH buffer is a
TRIS-CHES buffer.
15. The capillary column of claim 14 wherein the molarity of said
buffer is 100 mM.
16. The capillary column of claim 15 wherein the pH of said
composition is between about 8.0 and about 9.0.
17. A capillary column containing a dynamically cross-linked
composition comprising:
a) a capillary column having an interior cavity defined by a wall
with an inner surface;
b) a dynamically cross-linked composition filling said interior
cavity, said composition comprising:
i) about 1.0% polyethylene oxide;
ii) about 1.0% polyethylene glycol
iii) about 0.1% sodium dodecyl sulfate;
iv) about 100 mm TRIS-CHES buffer; and
v) about 1.0% ethylene glycol;
where the pH of the composition is between about 8.0 and about 9.0
and the viscosity of the composition is less than about 500
centipoise.
18. A method of performing capillary electrophoresis
comprising:
a) introducing an aliquot of a sample containing constituents to be
separated into a capillary column comprising a dynamically
cross-linked composition, the capillary column comprising:
i) a capillary column having an interior cavity defined by a wall
with an inner surface; and
ii) a dynamically cross-linked composition filling said interior
cavity, said composition comprising:
(a) between about 0.1% and about 1.5% polyethylene oxide;
(b) between about 0.0% and less than about 2.0% polyethylene
glycol;
(c) between about 0.0% and about 2.0% of a surfactant;
(d) between about 0.0M and about 1.0M of a pH buffer; and
(e) between about 0.0% and about 99% of a polyol; where the pH of
the composition is between about 2.0 and about 10.00 and the
viscosity of the composition is less than about 4,000
centipoise;
b) applying an electric field of at least about 10 volts per
centimeter to the capillary column;
c) separating the sample into its constituent parts; and
d) detecting the constituents of the sample.
19. The method of claim 18 wherein said sample comprises
surfactant-protein complexes.
20. The method of claim 19 wherein said surfactant of said
surfactant-protein complexes is selected from the group consisting
of sodium-dodecyl sulphate, decyl sulphate, polyoxyethylene ethers,
polyoxyethylenesorbitans, deoxycholate, cetyltrimethylammonium
bromide and cetylpyridinium chloride.
21. The method of claim 18 wherein said sample comprises sodium
dodecyl sulphate-protein complexes.
22. The method of claim 18 wherein said detecting is selected from
the group consisting of UV spectrophotometry, radioactive detecting
of fluorescence detecting.
23. The method of claim 19 wherein said detecting is UV
spectrophotometry at UV wavelengths in the range of 195-350 nm.
24. The method of claim 18 further comprising a layer of a coating
material on said inner surface of said wall.
25. The method of claim 18 wherein said capillary is constructed of
a material selected from the group consisting of glass, alumina,
beryllia, fused silica and TEFLON.
26. The method of claim 18 wherein said capillary is constructed of
fused silica.
27. The method of claim 18 wherein the internal diameter of said
capillary is between about 2 .mu.m and about 2000 .mu.m.
28. The method of claim 18 wherein said composition comprises about
1.0% polyethylene oxide.
29. The method of claim 18 wherein said composition comprises about
1.0% polyethylene glycol.
30. The method of claim 18 wherein said surfactant is selected from
the group consisting of sodium-dodecyl sulphate, decyl-sulphate,
polyoxyethylene ethers, polyoxyethylenesorbitans, deoxycholate,
cetyltrimethylammonium bromide and cetylpyridinium chloride.
31. The method of claim 18 wherein said surfactant is sodium
dodecyl sulphate.
32. The method of claim 18 wherein said composition comprises about
0.1% sodium dodecyl sulphate.
33. The method of claim 18 wherein said pH buffer is ultra-violet
light transparent.
34. The method of claim 18 wherein said pH buffer comprises
zwitterionic buffers.
35. The method of claim 18 wherein said pH buffer is selected from
the group consisting of 2-(N-morpholine) ethanesulfonic acid,
N-(2-acetamido) iminodiacetic acid,
piperazine-N,N'-bis(2-ethanesulfonic acid,
N-(2-acetamido)-2-aminoethanesulfonic acid, (2-aminoethyl)
trimethyl-ammonium chloride hydrochloride,
N,N-bis(2-hydroxy-ethyl)-2-aminoethane sulfonic acid,
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid,
trishydroxymethyl amino methane,
N-tris(hydroxylmethyl)methylglycine,
N,N-bis(2-hydroxyethyl)-glycine, 2-(N-cyclohexylamino)
ethane-sulfonic acid and mixtures of the foregoing.
36. The method of claim 18 wherein said pH buffer is a TRIS-CHES
buffer.
37. The method of claim 36 wherein the molarity of said buffer is
100 mM.
38. The method of claim 18 wherein the pH of said composition is
between about 8.0 and about 9.0.
39. A method of performing capillary electrophoresis on samples
comprising sodium-dodecyl sulphate-protein complexes comprising
a) introducing an aliquot of a sample comprising sodium-dodecyl
sulphate-protein complexes into a capillary column comprising a
dynamically cross-linked composition, the capillary column
comprising
i) a capillary column having an interior cavity defined by a wall
with an inner surface, and
ii) a dynamically cross-linked composition filling said interior
cavity, said composition comprising
(a) about 1.0% polyethylene oxide;
(b) about 1.0% polyethylene glycol;
(c) about 0.1% sodium dodecyl sulphate;
(d) about 100 mM TRIS-CHES buffer; and
(e) about 1.0% ethylene oxide:
where the pH of the composition is between about 8.0 and about 9.0
and the viscosity of the composition is less than about 500
centipoise;
b) applying an electrofield of at least about 10 volts per
centimeter to the capillary column;
c) separating the complexes; and
d) detecting the complexes.
Description
FIELD OF THE INVENTION
The present invention is directed to the analysis of samples in
general and in particular to the analysis of proteinaceous
materials using capillary electrophoretic techniques. In a
particular embodiment, the invention is directed to a capillary
column containing a dynamically cross-linked composition useful in
the analysis of surfactant:proteinaceous material complexes by
capillary electrophoresis.
BACKGROUND OF THE INVENTION
Capillary gel electrophoresis is one of the most widely used
separation techniques in the biologically-related sciences.
Molecular species such as proteins, peptides, nucleic acids, and
oligonucleotides are separated by causing the species to migrate in
a buffer solution under the influence of an electric field. The
buffer solution normally is used in conjunction with a low to
moderate concentration of an appropriate gelling agent such as
agarose or polyacrylamide to minimize the occurrence of mixing of
the species being separated. Two primary separating mechanisms
exist: a) separations based on differences in the effective charge
of the species; and b) separations based on molecular size.
The first of these mechanisms is generally limited to low or
moderate molecular weight materials, such as small oligonucleotides
(about 1 to about 50 nucleotides in length). This is because there
is typically an insignificant difference between the effective
charges of high molecular weight materials, making the task of
separation difficult or impossible.
Separations based on molecular size are generally referred to as
molecular "sieving". Molecular sieving utilizes gel matrices having
controlled pore sizes as the separating medium. The separation
results from the relative abilities of the different size molecular
species to penetrate through the gel matrix; smaller molecules move
more quickly than larger molecules through a gel of a given pore
size.
Medium-to-high molecular weight oligonucleotides (greater than
about 50 nucleotides in lenght), polypeptides, and proteins are
commonly separated by molecular sieving electrophoresis. Proteins
are heteropolyelectrolitic (i.e. an approximate equivalent number
of negative charged and positive charged moieties where the overall
molecule has a net neutral charge). As such, proteins become
charged molecules as they transit a charged capillay column.
Accordingly, in order to separate proteinaceous materials based
upon the size of the molecules, these materials must have the same
effective charge to mass ratio as they traverse the capillary
column.
Achieving the same effective charge to mass ratio is commonly
accomplished by treating the proteinaceous materials with a
surfactant, such as sodium dodecyl sulphate ("SDS"), and utilizing
a polyacrylamide gel material as the seiving medium. Such a
procedure is referred to as sodium dodecyl sulphate polyacrylamide
gel electrophoresis ("SDS-PAGE"). See, for example, Gel
Electrophoresis of Proteins: A Practiced Approach (Second Ed). B.
D. Harnes & D. Rickwood, Eds. IRL Press, Oxford University
Press, 1990. See also, New Directions in Electrochoretic Methods.
T. W. Jorgenson & M. Phillips, Eds. published by American
Chemical Society, Washington, D.C. 1987. Both of these references
are incorporated fully herein by reference.
A surfactant, such as SDS, comprises a hydrophobic (water-hating)
"tail" and a hydrophillic (water-loving) "head." Thus, a surfactant
interacts with a protein species via hydrophobic interactions
between the hydrophobic "tail" of the surfactant and the protein
species. Upon ionization, the hydrophillic "head" of the surfactant
molecules surrounding the protein species become negatively
charged, positively charged, or remain neutral; upon ionization,
SDS becomes negatively charged. Accordingly, an SDS:protein complex
has a uniform charge distribution, and such a complex can then be
separated based upon size relative to the pore-size distribution
throughout the gel matrix.
Commercially available capillary electrophoresis instruments the
P/ACE.TM. high performance capillary electrophoresis system
(Beckman Instruments, Inc., Fullerton, Calif., U.S.A.), utilize a
detection system based upon ultra-violet ("UV") light absorption.
While UV detection of SDS-protein complexes in polyacrylamide
filled capillaries is possible, such detection is limited to a
specific wavelength detection of about 250 nm and higher. This is
because of the high UV absorbance associated with both crosslinked
and uncrosslinked polyacrylamide gels.
Such dection limitations are a distinct disadvantage particularly
with respect to the analysis of proteins. This is because proteins
absorb UV light very strongly at 214 nm, due to peptide bonds
within proteins. Thus, UV detection of proteins should be conducted
at about 214 nm. However, because of the 250 nm and higher
detection limitations created by the use of polyacrylamide gels,
the sensitivity and selectivity of UV detection of proteinaceous
materials using polyacrylamide-based gel systems is limited.
Accordingly, UV detection of surfactant:proteinaceous materials
would be greatly improved if on-column detection was conducted at
lower UV wavelengths. This, in light of the foregoing, requires
molecular sieving materials that do not suffer the drawbacks of
polyacrylamide gels.
SUMMARY OF THE INVENTION
Disclosed herein are capillary columns containing dynamically
cross-linked compositions and which are applicable to the analysis
of surfactant:proteinaceous material complexes by capillary
electrophoresis. In a preferred embodiment, the dynamically
cross-linked compositions comprise between about 0.01% and about
1.5% polyethylene oxide; between about 0.0% and less than about
2.00% polyethylene glycol; between about 0.0% and about 2.0% of a
surfactant; between about 0.0% and about 99.0% of a polyol; and
between about 0.0M and 1.0M of a pH buffer, where the pH of the
composition is between about 2.0 and about 10.0. Preferably, the
viscosity of the dynamically cross-linked composition is less than
about 4,000 centipoise, more preferably less than about 500
centipoise, and preferably about 150 centipoise. The compositions
are particularly useful in the analysis of SDS-protein complexes
using UV detection-based capillary electrophoretic systems,
although other detection systems based upon, e.g., radioactive or
fluorescence detection, are equally applicable. The capillary
columns can be either untreated or coated with a conventional
coating material.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings are presented for the purpose of reference
in conjunction with the Detailed Description of Preferred
Embodiments of the Invention.
FIG. 1 is an electropherogram of the separation of low molecular
weight SDS-protein standard mixture using a 1.0%PEO/1.0%PEG
dynamically cross-linked composition;
FIG. 2 is an electropherogram of the separation of high molecular
weight SDS-protein standard mixture using the dynamically
cross-linked composition of FIG. 1; and
FIG. 3 is a calibration curve of the low-and high-molecular weight
SDS-proteins of FIGS. 1 and 2 based upon the relative migration
times thereof vis-a-vis an internal standard, Orange G.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Disclosed herein are capillary columns containing dynamically
cross-linked compositions for use in, most preferably, the analysis
of surfactant:proteinaceous material complexes using capillary
electrophoretic techniques.
As used herein, the phrase "dynamically cross-linked composition"
means a gel-like solution having a three-dimensional network of
polymer chains held together by hydrogen bonding and dispersed in a
liquid phase. The dynamically cross-linked composition is a viscous
liquid having a sufficient structure for a degree of rigidity which
allows for, inter alia. molecular sieving based upon the size of
the materials to be separated. Preferably, the viscosity of the
composition is less than about 4,000 centipoise, and more
preferably less than about 500 centipoise. When the composition
does not include PEG, it is a linear, as opposed to cross-linked,
composition. PEG is preferably added to the composition to
dynamically cross-link the PEO. Accordingly, "dynamically
cross-linked composition," as that phrase is used herein, includes
linear compositions such as compositions not including PEG as
described above.
As used herein, the term "capillary column" means a capillary
comprising an interior cavity defined by a wall with an inner
surface, where the internal diameter range of the capillary is
between about 2 .mu.m and 2000 .mu.m. If the detection system of
the capillary electrophoretic system is based upon UV absorbance,
then the capillary is preferably made of a UV transparent material,
such as, for example, glass or fused silica, with fused silica
being most preferred. If the detection system of the capillary
electrophoretic system is based upon, e.g. radioactive detection of
fluorescence detection, then the capillary is preferably made of a
material conducive to such systems. Alumina, beryillium,
TEFLON.TM.-coated materials, glass and fused silica are exemplary
materials. The capillary column should be capable of withstanding a
wide range of applied electrophoretic fields of between about 10
volts per centimeter ("V/cm"), up to about 1000V/cm. The capillary
column may be coated on the outside (using, e.g., a polyamide
material) for ease of handling. The inner wall of the capillary may
be untreated or coated with a coating material which is available
or known to those in the art. An example of a preferred coating
material is disclosed in U.S. Pat. No. 5,098,539, which is
incorporated fully herein by reference. Preferably, the internal
diameter of the capillary column is between about 2 .mu.m and 2000
.mu.m and most preferably about 100 .mu.m.
The term "UV transparent" as used herein, means having negligible
absorbance throughout the UV wavelength range of between about 195
nm and about 350 nm.
As used herein, the term "surfactant" is a substance having
hydrophobic and hydrophillic properties, and exhibiting either a
negative charge, a positive charge or neutral charge upon
ionization. The hydrophobic portion of the surfactant is capable of
interacting with a proteinaceous material via hydrophobic
interactions such that the material is surrounded by the
hydrophilic portion of the surfactant. Representative anionic
surfactants include, for example, sodium-dodecyl sulphate ("SDS"),
decyl-sulphate, and deoxycholate. Representative cationic
surfactants include, for example cetyltrimethylammonium bromide
("CTAB") and cetylpyridinium chloride ("CPC"). Representative
non-ionic surfactants include, for example, polyoxyethylene ethers
such as Triton X 100.TM. and Triton DF-16.TM., and
polyoxyethylenesorbitans such as BRIJ-35.TM., the TWEEN.TM.
surfactants, and LUBROL W.TM.. All of the foregoing
trademark-designated surfactants are available from Sigma Chemical
Co., St Louis, Mo. Of the surfactants, anionic surfactants are
preferred when used in conjunction with untreated fused silica
columns. Most preferably, the surfactant is SDS.
As used herein, the term "proteinaceous material" means, proteins
(both natural and those derived via recombinant nucleic acid
technology), peptides, polypeptides, nucleic acids and
oligonucleotides. It is to be understood that while the disclosed
dynamically cross-linked composition finds particular applicability
in the analysis of proteinaceous materials, and in particular
proteins, the disclosure is not limited to such materials. Thus,
the disclosed dynamically cross-linked composition can be utilized
for the analysis of other materials capable of being analyzed by
capillary electrophoretic techniques. Because proteinaceous
materials as defined herein can be charged upon ionization, it is
to be understood that the dynamically cross-linked composition need
not incorporate a surfactant therein. For example, deoxyribonucleic
acid ("DNA") molecules have the same charge-to-mass ratio;
therefore, a surfactant is not required to achieve this result.
However, it is preferred that the composition include a surfactant
for the analysis of proteinaceous materials.
The dynamically cross-linked composition comprises the following
materials: i) between about 0.01% and about 1.5% polyethylene oxide
("PEO"); ii) between about 0.0% and less than about 2.0%
polyethylene glycol ("PEG"); iii) between about 0.0% and about 2.0%
of a surfactant; iv) between about 0.0% and about 99% of a polyol;
and v) between about 0.0M and about 1.0M of a pH buffer, where the
composition has a pH of between about 2.0 and about 10.0, and the
viscosity of the composition is less than about 4,000
centipoise.
In a particulalry preferred embodiment, the dynamically
cross-linked composition comprises about 1.0% PEO; about 1.0% PEG;
about 0.1% of the same surfactant used in the formation of the
surfactant:proteinaceous material complex; and 100 mM of a pH
buffer, where the composition has a pH of between about 8.0 and
about 9.0, and a viscosity of less than about 500 centipoise.
Uncoated, or untreated, capillary columns can be utilized in
conjunction with the compositions. When uncoated capillary columns
are used, the dynamically cross-linked composition further
comprises between about 0.01% and about 99.00% of a polyol. The
polyol, in effect, circulates throughout the polymer to "coat" the
locations on the inner wall of the capillary which are not covered
by the dynamically cross-linked composition. Representative polyols
include, but are not limited to, ethylene glycol and glycerol. A
preferred polyol is ethylene glycol. Most preferably the
composition comprises about 1.0% of ethylene glycol. It is to be
understood that the composition can (and preferably does) comprise
polyol even when used in conjunction with coated capillary
columns.
When the detection system of the capillary electrophoretic system
is based upon UV absorbance, the pH buffer should be UV
transparent. Exemplary UV transparent buffers include, for example,
the so-called "Good" buffers (see Good, N. E. et al "Hydrogen Ion
Buffers for Biological Research" Biochemistry 5/2: 467-477 (1966)
which is incorporated herein by reference). The Good buffers can be
described as being zwitterionic buffers covering the range of
pK.sub.a from 6.15 to 8.75, and include 2-(N-morpholine)
ethanesulfonic acid ("MES"), N-(2-acetamido) iminodiacetic acid
("ADA"), piperazine. N,N'-bis(2-ethanesulfonic acid ("PIPES"),
n-(2-acetamido)-2-aminoethanesulfonic acid ("ACES"), (2-aminoethyl)
trimethyl-ammonium chloride hydrochloride ("Cholamine"),
N,N-bis(2-hydroxy-ethyl) 2-aminoethane sulfonic acid
("TES"),N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid
("HEPES"), tris-hydroxymethyl amino methane ("TRIS"),
N-tris(hydroxyl-methyl)methylglycine ("Tricine"),
N,N-bis(2-hydroxyethyl)-glycine ("Bicine"), 2-(N-cyclohexylamino)
ethane-sulfonic acid ("CHES"), and mixtures of the foregoing. A
particularly preferred pH buffer for UV detection purposes is
TRIS-CHES.
While the foregoing buffers are preferred for use in conjunction
with UV detection, it is to be understood that such buffers can
also be utilized with, e.g., radioactive detection or fluorescent
detection instruments. Additionally, when UV detection is not
utilized, buffers that include constituents having aromatic rings
or peptide bonds can be utilized as the pH buffer. TRIS-histidine
is an example of such a buffer.
The pH of the buffer is principally selected with respect to the
type of surfactant utilized. For cationic surfactants, the pH of
the buffer should be in the acidic range (i.e. between about 2.0
and 5.0); for anionic surfactants, the pH of the buffer should be
in the alkaline range (i.e. between about 8.0 and 10.0); for
nonionic surfactants, the pH of the buffer is between about 5.0 and
8.0. With respect to the preferred SDS surfactant, a most preferred
pH is about 8.8.
For UV detection-based analysis of proteinaceous materials using
untreated capillary columns, a particularly preferred embodiment of
the dynamically cross-linked composition comprises about 1.0% PEO;
about 1.0% PEG; about 0.1% SDS; about 1.0% ethylene glycol; and
about 100 mM TRIS-CHES buffer, pH 8.8, and a viscosity of less than
about 500 centipoise.
Capillary electrophoresis using the dynamically cross-linked
composition disclosed above includes the steps of introducing an
aliquot of a sample containing constituents to be separated into
the column of the invention, applying an electric field of at least
about 10 V/cm to the column, allowing a current of between about
1.0 to about 100 microampers(.mu.A) to pass through the column, and
detecting the constituents of the sample as they migrate past an
on-line detector. Introduction of the samples can be accomplished
by the electrokinetic injection method or pressure injection;
preferably, pressure injection is utilized. The electric field may
be a continuous or pulsed electric field; those skilled in the art
will appreciate the distinction between these types of fields.
Preferably, a continuous electric field is utilized.
The capillary "running buffer" is dependent principally on the
detection system. For UV detection, the running buffer should be UV
transparent. Preferably, the running buffer is the same buffer
utilized in the dynamically cross-linked composition. When the
composition does not include a pH buffer, the running buffer is
selected based upon the detection system, as indicated above.
Accordingly, the pH buffers disclosed above can be utilized for the
running buffer.
The dynamically cross-linked composition containing capillary
columns are particularly suited for the analysis of proteinaceous
materials over approximately 50 to 100 runs. As noted, the
capillary column can be either coated or untreated. With respect to
untreated columns, a deactivation solution should be passed through
the capillary after approximately every 10 analytical runs. As used
herein, the term "deactivation solution" is a solution which
deactivates the surface of the untreated capillary column. For
example, with fused silica, the SiO groups which occupy the inner
surface of the capillary wall during electrophoresis should be
converted to SiOH groups via the deactivation solution. A most
preferred deactivation solution is 1M HCl.
The analysis of proteinaceous materials can be made at UV
wavelengths of 214 nm using the disclosed composition. This is a
distinct advantage over polyacrylamide gels. Additionally, the
disclosed dynamically cross-linked polymer can be used for
quantitative analysis of, e.g., proteinaceous materials over large
molecular weight ranges. As those in the art appreciate, such
calibration plots for polyacrylamide gels is quite difficult to
achieve, thus heretofore making quantative analysis evasive.
The following examples are presented for illustrative purposes only
and are not intended, nor should they be construed to be, a
limitation on the foregoing disclosure or the claims to follow.
EXAMPLES
Capillary electrophoresis of samples described in the following
Examples was performed on a Beckman Instruments, Inc. PACE.TM. high
performance capillary electrophoresis system. This system contains
built-in 200, 214, 254, 260, 280 and 415 nm narrow-band filters for
on-line detection. The detection window was located approximately
7.0 cm from the column outlet and 40 cm from the column inlet.
Samples were placed on the inlet tray of the above-referenced
capillary electrophoresis system. Samples were automatically
injected into the dynamically cross-linked polymer capillary column
by utilizing the pressure injection mode for 1-90 seconds.
Capillary columns, coated in accordance with the disclosure of U.S.
Pat. No. 5,098,539 had a column length of 47 cm, an effective
column length of 40 cm, and an internal diameter of 100 .mu.m.
Electric field strength was 300 V/cm (14.1 KV for 47 cm); current
utilized was 25-30 .mu.A.
EXAMPLE I
Material Preparation
A. Running Buffer Solution
A 0.5M TRIS-CHES buffer (pH 8.8) was prepared by dissolving 12.1 g
TRIS (ICN Biochemicals, Irvine, Calif.; Product No. 819620) in 100
ml deionized water. Solid CHES (ICN, Product No. 101434) was added
with continuous stirring until a pH of 8.8 was achieved. A final
volume of 200 ml was achieved using deionized water.
B. Model Protein Sample Preparation
A 0.3M TRIS sample buffer was adjusted with 1:1 HCl to pH 6.6,
followed by addition of 10% SDS thereto, as follows. 36.3 g TRIS
was dissolved in 500 ml deionized water, followed by addition of
1:1 diluted HCl (VWR, San Francisco, Calif., Product No. VW3110-3)
with continuous stirring and pH monitoring until a pH of 6.6 was
achieved. 100 g SDS (ICN, Product No. 811034) was added with
continuous stirring and a final volume of 1000 ml was achieved
using deionized water.
Model Protein SDS-protein standards were as follows:
1) Bio-Rad Labs, Richmond, Calif., Catalogue No. 161-0303
(molecular weight range of 14,000-97,000 Daltons:Lysozyme; Soybean
trypsin inhibitor; Carbonic anhydrase; Ovalbumin; Bovine Serum
albumin; Phosphorylase b.); and
2) Bio-Rad Labs, Catalogue No. 161-0304 (molecular weight upper
limit of 200,000 Daltons: Ovalbumin; Bovine Serum albumin;
Phosphorylase b; Beta-galactosidase; Myosin).
Orange G.TM. dye (7-hydroxy-8-phenylazo-1,3-napthalenedisulfonic
acid; Sigma Chemical Corp., St. Louis, Mo. Product No. 03756) was
utilized as an internal standard. A 0.1% solution, in deionized
water, was utilized for the Examples.
Protein disulfide linkages were broken using 2-mercaptoethanol
(ICN, Product No. 190242). As those skilled in the art appreciate,
in the presence of a surfactant and a reducing agent, most
multichain proteins will bind the surfactant to a constant value
and the disulfide bonds will be broken by the reducing agent. Thus,
the secondary structure of the protein will be lost and the
surfactant-protein complex is assumed to adopt a random coil
confirmation. Accordingly, proteins treated in this manner have a
uniform shape and identical mass to charge ratios. Thus, it should
be appreciated that any reducing agent is applicable, such as, for
example, dithiothreitol, (DTT) and mercaptoethanol.
Samples were prepared by admixing 0.1 to 0.2 mg of the Model
Proteins (i.e. 20 .mu.l of the Bio Rad materials), 40.mu.l of the
sample buffer; 10 .mu.l of the Orange G solution; and 5 .mu.l of
2-mercaptoethanol. A final solution volume of 125 .mu.l was
achieved using deionized water. This final mixture was boiled in a
water bath (100.degree. C.) for 15 min. in a closed microcentrifuge
vial, then cooled on ice water for 2 min. before introduction into
the capillary column.
C. Dynamically Cross-Linked Composition
10 g of PEO (MW 900,000; Aldrich Chemical Co., Milwaukee, Wis.,
Product No. 18,945-6) and 10 g PEG (mw 35,000; Fluka, Ronkonkoma,
N.Y., Product No. 81310) were mixed with 10 ml ethylene glycol
(VWR, Product No. EM-EX0564-1) and 50 ml deionized water for 10
min. in a 1.2 liter wide-neck flask. Thereafter, 700 ml of
deionized water was added thereto, followed by stirring with a
magnetic bar at 50.degree. C. for three hours. After this, 200 ml
of Running Buffer was added thereto, followed by stirring for one
hour. 10 ml of 10% SDS (1.0 g SDS completely dissolved in 10 ml
deionized water) was then added, followed by addition of deionized
water to achieve a final volume of 1000 ml, and overnight stirring
at room temperature. Prior to use, the dynamically cross-linked
composition was sonicated for 5 min. to remove air bubbles.
The viscosity of the dynamically cross-linked composition was
determined using an ELV-8.TM. viscometer, Col-Parmer, Inc.,
Chicago, Ill., using 100 ml of the composition. Viscosity was
determined to be 150 centipoise at 25.degree. C. relative to equal
volumes of distilled water (20 centipoise) and glycerol (1100
centipoise) measured at the same temperature.
EXAMPLE II
Analysis of Low Molecular Weight SDS-Protein Standard Mixture
Excellent separation and specificity was achieved in less than 20
minutes for the analysis of the low molecular weight standards
under the parameters set forth above. FIG. 1 provides the
electropherogram results of such analysis, where "O.G." is the
Orange G internal standard; "1" is Lysozyme "2" is Soybean trypsin
inhibitor; "3" is Carbonic anhydrase; "4" is Ovalbumin; "5" is
Bovine serum albumin; and "6" is Phosphorylase b.
EXAMPLE III
Analysis of High Molecular Weight SDS-Protein Standard Mixture
Excellent separation and specificity was achieved in less than 20
minutes for the analysis of high molecular weight standards under
the parameters set forth above. FIG. 2 provides the
electropherogram results of such analysis where OG is as defined in
Example II; "1" is Ovalbumin; "2" is Bovine serum albumin; "3" is
Phosphorylase b; "4" is Beta-galactosidase; and "5" is Myosin.
EXAMPLE IV
Calibration Curve
As noted, SDS-PAGE protocols do not readily lend themselves to the
production of calibration curves over a wide range of molecular
weights. As should be appreciated, such curves can be utilized for,
inter alia. the determination of the molecular weight of an unknown
sample. Because the disclosed dynamically cross-linked composition
can be used to efficiently separate low-to-high molecular weight
proteins (i.e. 10,000 to 200,000 Daltons), such a calibration curve
can be generated.
FIG. 3 provides such a calibration curve, where each point
represents the migration time of each SDS-protein complex relative
to the internal standard. In FIG. 3, "1" is Lysozyme; "2" is
Soybean trypsin inhibitor; "3" is Carbonic anhydrase; "4" is
Ovalbumin; "5" is Bovine serum albumin; "6" is Phosphorylase b; "7"
is Beta-galactosidase; and "8" is Myosin. The Relative Standard
Deviation (RSD) for the calibration curve of FIG. 3 is 0.98.
Such a calibration curve has a myriad of applicable uses,
including, but not limited to, determination of unknown protein
molecular weights, and comparative analysis between
natural-proteins and proteins derived via recombinant DNA
technologies.
While the foregoing capillary column containing the dynamically
cross-linked composition and method of use thereof has been
described in considerable detail, it is to be understood that the
invention is not limited to preferred or disclosed embodiments. The
invention is not to be construed as limited in its applicability to
the particular high performance capillary electrophoretic system
described. Modifications and changes that are within the purview of
those skilled in the art are intended to fall within the scope of
the following claims.
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